Leishmaniasis, a vector-borne parasitic disease, is caused by obligate intramacrophage protozoa. It is characterized by diversity and complexity. It presents itself with a wide range of clinical forms. However, there are mainly 4 clinical forms. The Visceral Leishmaniasis (VL), also known as kala azar, is the most severe form of the disease, which, if untreated, has a mortality rate of almost 100%. The Cutaneous Leishmaniasis (CL) produces skin ulcers on the exposed parts of the body, such as the face, arms and legs. The number of ulcers may vary from 1 to as many as 200 in some cases, causing serious disability and leaving the patient permanently scarred. The third form is Mucocutaneous Leishmaniasis (MCL), or espundia. It can lead to extensive and disfiguring destruction of mucous membranes of the nose, mouth and throat cavities and can involve even the cartilage. The cutaneous form may lead to disseminated form, known as Diffuse Cutaneous Leishmaniasis (DCL). Leishmaniasis is caused by a total of about 21 species, which are transmitted by about 30 species of phlebotomine sandflies [Herwaldt B L., 1999].
The leishmaniasis are presently endemic in 88 countries on five continents, Africa, Asia, Europe, North America and South America, and a total of 350 million people are at risk of infection. It is estimated that worldwide 12 million people are affected by leishmaniasis; this figure includes cases with overt disease and those with no apparent symptoms. Of the 1.5-2 million new cases estimated to occur annually, only 600 000 are officially declared. Of the 500 000 new cases of VL, which occur annually, 90%, are in five developing countries: Bangladesh, Brazil, India, Nepal and Sudan. About 90% of all cases of MCL occur in Bolivia, Brazil and Peru and 90% of all cases of CL occur in Afghanistan, Brazil, Iran, Peru, Saudi Arabia and Syria, with 1-1.5 million new cases reported annually worldwide. The geographical distribution of leishmaniasis is limited by the distribution of the sandfly, its susceptibility to cold climates, its tendency to take blood from humans or animals only and its capacity to support the internal development of specific species of Leishmania [Desjeux P 2001].
Since 1993, regions that are Leishmania-endemic have expanded significantly, accompanied by a sharp increase in the number of recorded cases of the disease. The geographic spread is due to factors related mostly to development. These include massive rural-urban migration and agro-industrial projects that bring non-immune urban dwellers into endemic rural areas. Man-made projects with environmental impact, like dams, irrigation systems and wells, as well as deforestation, also contribute to the spread of leishmaniasis. AIDS and other immunosuppressive conditions increase the risk of Leishmania-infected people developing visceral illness [Desjeux P 2001, Paredes R et al., 1997].
VL is primarily caused by L. donovani in the Indian subcontinent and Africa, Leishmania infantum in Mediterranean region and Leishmania chagasi in the new world; of these species Leishmania chagasi and Leishmania infantum are closely related. Although, all the above species cause VL they are genetically different from each other. The data obtained by Cupolillo E et al., [1994] using numerical zymotaxonomy showed that L. chagasi, the new world visceralising species is similar to the old world L. infantum. The Zymodeme, serodeme, quantitative comparisons of nuclear DNA fragment patterns all indicates that L. chagasi and L. Infantum are closely related and may represent the same species. Also, the study by Beverley S. M. et al., [1987] based on nuclear DNA restriction fragment patterns reveals that, the L. chagasi and L. infantum are similar and as closely related to each other as two random individuals from the same population and L. donovani is different from these two species. In another study using analysis of repetitive DNA sequence by Piarroux R et al., [1995] it was observed that, amongst the leishmania causing VL, L. donovani isolated from foci in which human beings are the main reservoirs clustered in an independent branch and by contrast, L. infantum and L. chagasi are canine parasites that rarely infect human beings and thus are different. A recent study by Mauricio I. L et al., [1999] using three different approaches at different levels of resolution to explore the genetic information from leishmania species reveals a substantial amount of diversity within L. donovani complex. Further, RAPD had grouped L. donovani strains according to the geographical origins, specifically Indian and Kenyan, showing a substantial divergence within taxon.
Genetic diversity is not only common for L. donovani, even in L. major which causes cutaneous leishmaniasis, strains isolated from the same geographical area show minor chromosomal size polymorphisms in their molecular karyotypes whereas strains from different geographical areas show more significant differences suggesting that the genomes of species of leishmania are quite plastic and that chromosomal rearrangements occurs during the evolution of various species [Samaras N et al., 1987]. Currently a WHO sponsored genome mapping project on L. major is underway. Although it has been argued that the genome map of one strain would be applicable to another, there is very little evidence to substantiate this claim. Indeed, it is known that differences in gene copy number and organization differ between L. donovani, L. chagasi, L. major and other species. Moreover, it is difficult to reconcile the great differences in clinical symptoms caused by different species with identical genotype [Ghosh S. S., et al., 1998]. For these reasons, it is necessary to characterize important genes, which have potential to be a diagnostic or vaccine or therapeutic candidate from different geographical regions. The assignment of the parasite species based alone on geographic location or the site of infection is not satisfactory. Accordingly, correct diagnosis and classification of pathogenic Leishmania isolate is essential to determine the clinical prognosis and a species-specific therapeutic approach [Marfurt J., et al., 2003]. One such potential gene studied widely across different species from different geographical region is Gp63 a glycolipid-anchored zinc protease of 63 kDa size [Webb J. R., et al., 1991; Steinkraus H B et al., 1993; Roberts S C et al., 1993].
In India, VL is a serious problem in Bihar, west Bengal and eastern Uttar Pradesh where, there is under-reporting of kala-azar (KA) and post kala-azar dermal leishmaniasis in women and children of 0-9 years of age. The recent epidemics in 1992 of VL killed more than 100,000 people in India and Sudan. Spraying of DDT helped control KA in India, however there are reports of the vector phlebotomus argentipes developing resistance. Also, lymphadenopathy, a major presenting feature in India raises the possibility of a new vector or a variant of the disease [Bora D., 1999].
The Post kala-azar dermal leishmaniasis (PKDL) is a sequel to KA in India and Sudan; the disease develops months to years after the patient recovery from KA. Cutaneous lesions characterize the disease and they demonstrate great variability, ranging from hypo-pigmented macules to erythematous papules and from nodules to plaques. As in leprosy, the wide clinical spectrum of PKDL reflects the immune response of the individual to the leishmania organism. Lesions may be numerous and persist for decades. Isolated parasites from the lesions are identical to those causing the original visceral disease.
The clinical and epidemiological findings in leishmaniases are not pathognomic and these can mimic with several endemic conditions such as malaria, tuberculosis, syphilis and fungal infections. Hence a laboratory diagnosis is required to confirm the clinical suspicion. The diagnostic tools used for each leishmanial syndrome viz. visceral, cutaneous, and mucocutaneous form, vary but the gold standard in each case remains the demonstration and isolation of the parasite from appropriate tissue [Singh S et al., 2003].
The clinical signs and symptoms are not enough to differentiate VL from other similar conditions such as malaria, tropical splenomegaly syndrome schistosomiasis or cirrhosis with portal hypertension, African trypanosomiasis, milliary tuberculosis, brucellosis, typhoid fever, bacterial endocarditis, histoplasmosis, malnutrition, lymphoma, and leukemia. Hence other diagnostic methods are required [Herwaldt B L, 1999; Davidson R N, 1998]. Amongst these the most specific and standard technique is parasitological demonstration or isolation of the causative agent. Marrow obtained from sternal or iliac crest puncture is a much safer but a painful method. The aspirates are smeared on the glass slide and stained with Romanowsky's stain to demonstrate the amastigote forms of the parasite. However; on culture it can give positive results in up to 80% of the cases. Lymph gland puncture gives positive results in 60% of the cases. Juice is extracted from any enlarged lymph gland and subjected to both direct examination and culture to give the best chance of diagnosis [Williams, J. E, 1995; Manson-Bahr PEC, 1987]. Primary isolation of L. donovani is made on solid Novy-MacNeal-Nicolle (NNN) medium having 20-30% rabbit blood or liquid Schneider's insect medium supplemented with 10% v/v foetal calf serum (FCS). Other suitable growth media can also be used particularly for maintaining the subcultures of the promastigotes using FCS or other supplements including human urine [Singh S et al., 2000]. Demonstration of the parasites in the spleen and liver is one of the most accurate methods available to determine leishmanial infections. Ninety percent of the active cases show parasites in splenic and liver aspirates. The smallest needle possible, preferably, 21-gauge (0.8 mm) should be used to minimize the risk of complications such as hemorrhage of the spleen [Williams, J. E, 1995]. Part of the splenic aspirate can be used to make smears for direct microscopic examination and the rest should be cultured. Liver biopsy material is less likely to demonstrate parasites on direct examination or on culture; however histological examination will show amastigotes in Kupffer cells in the portal system.
Occasional reports of finding the Leishmania parasites in blood in patients of Kala-azar from Kenya and India have been published. Blood in anticoagulant is centrifuged at 2000 g for 10 min and the cells from the buffy coat removed and used to prepare smears and inoculate cultures. The amastigotes can be found in and around Macrophage cells. The volume used in culture inoculation is important, 1-3 drops on NNN or Schneider's medium has given successful results [Manson-Bahr PEC, 1987].
The conventional microscopic methods are invasive and painful carrying risk of iatrogenic infections and fatal hemorrhages. Though demonstration of the amastigote form of parasite in the tissues is being used since its discovery as a parasitic disease in 1903, it is least sensitive and unable to detect occult and sub clinical infections. The sub clinical and latent form of infection has become a major concern in recent years, as these can flare up due to immune suppression such as in HIV infection and the infection can be transmitted through organ transplants. Serological diagnosis is based on the presence of specific humoral response as in cases of visceral leishmaniasis or cell mediated immune response in cases of cutaneous and mucocutaneous leishmaniasis, evoked by the immune system against the causative pathogen. There are ranges of serological methods available for the diagnosis of VL varying in accuracy and specificity. These included non-specific and specific tests. With on-going research newer better methods are continually becoming available.
The formol gel test is oldest serological test and has the advantage of being cheap and simple to perform. Serum obtained from about 5 ml of blood is mixed with one drop of 30% formaldehyde. A positive reaction is shown if the mixture solidifies and forms a white opaque precipitate within 20 minutes. A positive test cannot be detected until 3 months after infection and becomes negative 6 months after cure. The test is non-specific since it is based on detecting raised levels of IgG and IgM immunoglobulins which are also raised in other infections such as African trypanosomiasis, malaria and schistosomiasis etc., [WHO expert committee report, 1991]. Several other tests based on this principle had been in use in past but very rarely used these days [Singh S, 1999].
There are number of specific serological tests and all have variable sensitivity and specificity for disease diagnosis. Some of these tests include indirect haemagglutination (IHA), counter current immunoelectrophoresis (CCIEP), Immunodiffusion (ID) etc. but all these tests are cumbersome and lack sensitivity and specificity and hence not commonly used. Some more commonly used ones are described below.    1. Leishmanin Skin Test (LST): Delayed hypersensitivity is an important feature of cutaneous forms of human leishmaniasis and can be measured by the leishmanin test, also known as the Montenegro reaction. Leishmanin is a killed suspension of whole (0.5-1×107/ml) or disrupted (250 μg protein/ml) promastigotes in pyrogen-free phenol saline. No cross-reactions occur with chagas disease, but some cross-reactions are found with cases of glandular tuberculosis and lepromatous leprosy. Leishmanin Skin Test is usually used as an indicator of the prevalence of cutaneous and mucocutaneous Leishmaniasis in human and animal populations and successful cure of the visceral leishmaniasis [Singh S, 1999, Sassi A, et al., 1999]. During active kala-azar disease there will be no or negligible cell mediated immune response. However, the leishmanin antigen is not commercially available and no field study has been carried out in India.    2. Indirect fluorescent antibody test (IFAT): The Indirect fluorescent antibody test is one of the most sensitive tests available. The test is based on detecting antibodies, which are demonstrated in the very early stages of infection and undetectable six to nine months after cure. If the antibodies persist in low titers it is good indication of a probable relapse. Titers above 1/20 are significant and above 1/128 are diagnostic [Williams, J. E, 1995]. There is a possibility of a cross reaction with trypanosomal sera, however, this can be overcome by using leishmania amastigotes as the antigen instead of the promastigotes [Gari-Toussaint M, et al., 1994].    3. Agglutination test: The DAT is a highly specific and sensitive test. It is cheap and simple to perform making it ideal for both field and laboratory use. The antigen is prepared from promastigotes of L. donovani and test can be carried out on plasma, serum, blood spots and whole blood. For long time DAT remained first line diagnostic tool in resource poor countries. The method uses whole, stained promastigotes either as a suspension or in a freeze-dried form. The freeze-dried form is heat stable and facilitates the use of DAT in the field. However, the major disadvantage of DAT is the relative long incubation time of 18 h and the need for serial dilutions of blood or serum [Schallig H D et al., 2001]. Another major disadvantage of DAT is that it has no prognostic value for evaluating the parasitological cure of the disease, as the test may remain positive for several years after cure. Recently, Schoone et al [2001] have developed a fast agglutination-screening test for the rapid detection (<3 h) of anti-leishmania antibodies in serum samples and on blood collected on filter paper. The FAST utilizes only one serum dilution leading to qualitative results. The FAST offers the advantages of the DAT based on the freeze-dried antigen with respect to stability of the antigen, reproducibility, specificity and sensitivity.    4. Immunoblotting: Serodiagnosis using immunoblotting has been attempted and reported superior and stage specific. The various antigens expressed during the course of infection can also be documented. It also has an added advantage of permanent documentation. However, the technique is not user friendly and limited only to research laboratories [Herwaldt B L., 1999; Singh S, 1999; Schallig H D et al., 2001].    5. Antigen Detection: The detection of antigen in the patient's serum is complicated by the presence of high level of antibodies, circulating immune complexes, serum amyloid, rheumatoid factor and auto antibodies all of which may mask immunologically important antigenic determinants or competitively inhibit the binding of free antigen. Antigen detection test would, in principle provide better means of diagnosis of leishmaniasis. Since antigen levels are expected to broadly correlate with the parasite load, the antigen detection may be an ideal alternative to the antibody detection in immunocompromised patients, where antibody response is very poor. Though a few reports are published, no satisfactory antigen detection system is currently available [Senaldi G et al., 2001; Attar Z J et al., 2001]. Recently, a latex agglutination test (KATEX) for the detection of leishmanial antigens in the urine of patients with VL is developed. The results obtained with KATEX using samples collected from different foci of VL indicate that, the test works well regardless of the geographical origin of samples. The test had 100% specificity and sensitivity between 68-100% [Attar Z J et al., 2001]. Whether the test has applications for the detection of asymptomatic cases of VL and monitoring therapy is yet to be confirmed.    6. Enzyme linked immunosorbent assay (ELISA): The Enzyme Linked Immunosorbant Assay (ELISA) is a valuable tool in the serodiagnosis of leishmaniasis. The test is useful for laboratory analysis or field applications. The ELISA can be performed easily and is adaptable for use with purified or defined antigen. The antigens used in the design of immunodiagnostic tests for leishmaniasis have traditionally been derived from promastigotes that have been cultivated in vitro or from recombinant proteins, alteration of the antigen used for ELISA and DAT from the whole promastigote or soluble antigens to more specific and potential recombinant leishmanial and peptide antigens have improved VL diagnosis [Senaldi G et al., 2001]. Immunodiagnosis is greatly influenced by the antigen used. Several antigen molecules have recently been reported [Martin S K et al., 1998; Rajasekariah G H et al., 2001]. The excretory, secretary and metabolic antigens (Ld-ESM), released by L. donovani promastigotes into a protein-free medium were used for the serodiagnosis of VL by ELISA. The Ld-ESM has been found to be 100% specific and sensitive, the Positive Predictive Value was 99.99% and Negative Predictive Value was 95.45%. However further retrospective and prospective multisite evaluation is required to validate these findings [Schoone G J et al., 2001]. Lately, a variety of recombinant antigens have been developed, recently a gene related to the L. major gene B encoding a hydrophilic protein expressed on the surface of both promastigotes and amastigotes of L. major characterized by an amino acid repeating motif of 5.5 copies of a 14-amino acid sequence has been identified and shown to be expressed in L. donovani. The protein encoded by L. donovani gene B homologue contains up to 22 copies of a repetitive element in which 9 out of 14 residues are completely conserved between the two species. An ELISA using repetitive peptide sequence from L. donovani GBP and recombinant L. donovani GBP as solid-phase ligand was developed. However the limitations of this antigen are that it can be used for serodiagnosis of visceral leishmaniasis only in areas endemic for L. donovani but not for areas that are co-endemic for other Leishmania species and the specificity and sensitivity are not very high [Jensen A T et al 1999].
Raj et al., [1999] have developed another recombinant protein rORFF of L. infantum origin for diagnosis of VL in India. The ORFF protein is encoded in the LD1 locus of chromosome 35 of L. infantum, an ELISA with this antigen proved to be sensitive with as little as 5 ng of rORFF when performed with different groups of patients like confirmed VL, suspected VL, Intermittently treated endemic normal and non-endemic normal controls. Further the same patient groups were subjected to DAT using whole promastigote and ELISA using total soluble antigens. The ELISA using rORFF was found to be more sensitive than others. Although this antigen is highly sensitive (95 to 100%) and specific (>90%) for VL, it also was found to be positive in 40% cases of confirmed CL due to L. major or L. tropica. Further the test needs to be evaluated by others and its utility for the field diagnosis is yet to be studied. In a recent study conducted in Mediterranean VL where L. infantum is the causative agent, ten recombinant and purified leismania antigens have been compared using ELISA method by Maalej et al., [2003]. Of these recombinant antigens rgp63, a major surface antigen of leishmania which is not present on Trypanosoma cruzi or other kinetoplastids and rGBP had good performance but not very sensitive and specific for reliable diagnosis. It is suggested that the use of recombinant proteins from L. infantum rather than L. major would have yielded a better result.
A recombinant antigen developed by Burns et al., [1993] belonging to the kinesin family of motor proteins, recombinant K39 (rK39) has been shown to be specific for antibodies arising during VL caused by members of the L. donovani complex, which include Leishmania chagasi and L. infantum. This antigen, which is member of the kinesin family, encodes a protein with a repetitive epitope, consisting of 39 amino acid residues (K39) is highly sensitive and predictive of acute disease. The high anti-rK39 antibody titers have been demonstrated in VL patients but it shows no detectable anti-rK39 antibodies in cutaneous or mucocutaneous leishmaniasis. The antibody titers to this antigen directly correlate with active disease and have a tremendous potential as a means of monitoring chemotherapy and in predicting clinical relapse [Burns J M Jr et al., 1993; Singh S et al., 1995; Badaro R et al., 1996; Singh S et al., 2002; Maalej I A et al., 2003; The U.S. Pat. Nos. 5,411,865 and 5,719,263]. In addition rK39 ELISA, has a high predictive value for detecting VL in immunocompromised persons, like AIDS patients [Houghton R L et al., 1998]. This antigen is now commercially available in the form of antigen-impregnated nitrocellulose paper strips adapted for use under field conditions. The rK39 strip test has been found useful for the field diagnosis of kala-azar in India [Sundar S et al., 1998] however the same had markedly less sensitivity in Sudan [Zijlstra E E et al., 2001] and southern Europe. It is important to emphasize here that though the kinesin related antigen gene has been shown to be conserved in all visceralising species, but the same seems not to be case, because L. donovani is the causative agent of kala-azar in India as well as in Sudan, and in both the geographical regions they cause PKDL as a sequel to VL. But the observations of Zijlstra et al., [2001] that, the rK39 strip tests are less sensitive (only upto 67%) in Sudan and even in Southern Europe (only upto 71.4%) [Jelinek T et al., 1999] raise the valid doubt about the universal suitability of this antigen. One explanation often given for this variable sensitivity is that may be the antibody response elicited by different ethnic groups differs remarkably [Sundar S et al., 2002]. Alternatively, it could also be possible that, the antigenic gene itself varies notably from strain to strain and also in different geographical regions or a variant of this antigen exist and evades immune elucidation as a result the disease goes undetected in few cases when the existing rK39 from L. chagasi is used for the diagnosis. Further, of the 500,000 new case of VL, which occurs annually worldwide, more than 90% of are reported from India, Bangladesh, Southern Sudan, and northeast Brazil [Sundar S et al., 2002].
India harbors majority of VL cases in the world, and the kinesin related antigen was not yet characterized from the Indian isolates of Leishmania donovani. It is also evident that, L. chagasi has the animal reservoir while the L. donovani does not. It is possible that, the gene differs significantly from the L. chagasi and the characterization of this gene will also explain reasons for the poor sensitivity of rK39 strip test in certain geographical regions. In an attempt to solve this problem, we have cloned and characterized the kinesin gene from different strains isolated from two individuals infected by L. donovani belonging to the same geographical region in India. One strain MHOM/IN/DD8 was the well characterized WHO reference strain for India isolated in 1980 and the second strain MHOM/IN/KE16/1998 is a recent clinical isolate from a 10 year old female Kala-azar patient from Muzaffarpur, Bihar, India. This clearly explains that, it is the variation at the gene level that may have caused reduced sensitivity in certain geographical regions. Had this kinesin gene not been characterized from Indian isolates, this information of variation at the gene level never would have become known to the scientific community. This is evident from the ELISA results, as the mean titers for the antigen from MHOM/IN/DD8 is considerably lower than for the one of MHOM/IN/KE16/1998 and L. chagasi rK 39.
The applicants did extensive search of the US patent database with different key words to study the previous work done on the K39 immunodominant repeat antigen and 230 kD antigen. Discussed below are the few US patents by Reed on the subject concerned and the uniqueness of the applicant's antigen.
The U.S. Pat. No. 5,411,865 by Reed in May 2, 1995 teaches about the method of detecting anti-leishmania parasite antibodies. The compound disclosed a method for detecting anti-Leishmania parasite antibodies to a 230 kDa antigen present in Leishmania chagasi and Leishmania donovani which comprises obtaining a sample from an individual, contacting the sample with a recombinant K39 repeat regionantigen comprising the amino acid sequence as shown in SEQ ID NO: 3, and detecting the presence of anti-Leishmania parasite antibodies in the sample which bind to the recombinant K39 repeat regionantigen.
The U.S. Pat. No. 5,719,263 by Reed in Feb. 17, 1998 teaches about the 230 Kd antigen present in Leishmania species. The compound disclosed is an isolated 230 kD antigen that is present in Leishmania chagasi and Leishmania donovani, and isolated polypeptides comprising one or a plurality of K39 repeat antigens. Also disclosed are DNAs encoding the 230 kD antigen and the K39 repeat antigen, and vaccine compositions comprising the antigens.
The above disclosed 230 kDa antigen and the isolated polypeptide comprising the K39 repeats are reported to be not sensitive in certain geographical areas where VL is highly endemic and caused by L. donovani. However, it should be noted that, the K39 repeats has been characterized only from L. chagasi and not from L. donovani. The major burden in leishmaniasis throughout the world is caused by L. donovani. It is also evident from the reports that, the two species are genetically different. Hence, the applicants cloned and characterized K39 repeat immunodominant region from the Indian isolates of Leishmania donovani, which contributes significantly to the global VL burden.
The applicant's present invention discloses a 29 kDa and 26 kDa repeat antigen, which is present in leishmania species. The compounds disclosed are an isolated 29 kD and 26 kD antigens, which are characterized from the Indian isolates of Leishmania donovani. The reported antigens are entirely different from the Leishmania chagasi 230 kDa antigen. The antigen varies significantly, more than 60% in its predicted amino acid sequence to that of K39 repeat antigen from L. chagasi. Also disclosed are DNA encoding the 29 kD and 26 kD) antigen and therapeutic and vaccine compositions comprising the antigens.
The U.S. Pat. No. 5,912,166 by Reed, et al., in Jun. 15, 1999 teaches about compounds and methods for diagnosis of leishmaniasis infection. The compounds provided include polypeptides that contain at least an epitope of the Leishmania chagasi acidic ribosomal antigen LcP0, or a variant thereof. Such compounds are useful in a variety of immunoassays for detecting Leishmania infection and for identifying individuals with asymptomatic infections that are likely to progress to acute visceral leishmaniasis. The polypeptide compounds are further useful in vaccines and pharmaceutical compositions for preventing leishmaniasis.
However, the applicant's present invention does not deal with acidic ribosomal antigen LcPO.
The U.S. Pat. No. 6,638,517 by Reed, et al., in Oct. 28, 2003, Leishmania antigens for use in the therapy and diagnosis of leishmaniasis teaches compositions and methods for preventing, treating and detecting leishmaniasis and stimulating immune responses in patients. The compounds provided include polypeptides that contain an immunogenic portion of one or more Leishmania antigens, or a variant thereof. The patent also discloses vaccines and pharmaceutical compositions comprising such polypeptides, or polynucleotides encoding such polypeptides, are also provided and may be used, for example, for the prevention and therapy of leishmaniasis, as well as for the detection of Leishmania infection.
The compounds provided in the above patent utilize a sequence of L. major origin and a fusion construct from multiple leishmania antigens. These compounds are in no way similar to the applicant's antigen
The kinesin related antigen gene has been cloned and characterized from L. chagasi an South American visceralising species by Burns et al., [1993], this led to the development of rK39 antigen, a recombinant protein with 39 amino acid tandem repeats. This antigen from a new world visceralising species L. chagasi is reported not sensitive in some highly endemic regions for VL, Sudan and Southern Europe as it is on some other geographical areas, India, Bangladesh, Nepal etc., [Sundar et al., 2002] India carries the majority of the VL population in the global leishmaniasis burden. So, it is very important to have precise and well characterized tools for the diagnosis and other control measures. The applicants have characterized the kinesin related antigen at the sequence level to study, whether the sequence is similar to that of L. chagasi which is well characterized and to rule out the possibility of some VL and PKDL cases going undetected in India as those reported in Sudan. This study will also through light on the reasons for the varied response among the ethnic groups, which harbor this disease to the rK39 antigen of Burns et al., [1993]. In the present invention we found that, the sequence of our kinesin antigen significantly differs from that of the published reports of L. chagasi from which the rK39 of Burns et al, was derived. This invention also provides a method for detection of anti-leishmanial antibodies using the antigens derived from the Indian isolates of L. donovani. 
The study was important to analyze the response to the antigen in the various ethnic groups in India. It is possible that the antibody levels in the diseased people of India may be due to either the strains being different or it is possible that the various ethnic groups respond to the Leishmania antigen in different manner.